Nucleic acid hybridization is a well known procedure for investigating the identity of nucleic acids. Hybridization is based on complementary base pairing. When single-stranded nucleic acids are incubated in solution, complementary base sequences pair to form double-stranded hybrid molecules. These molecules can be separated when desired by denaturation.
Also known are nucleic acid probe assays which can be used to assay a specimen for the presence of a predetermined (also known as a target) nucleic acid for diagnosis of disease, genetic defects, genetic engineering or characterization, or for testing blood, food or other materials for contamination or other medical or research purposes see, for example, U.S. Pat. No. 4,358,535 (issued Nov. 9, 1982 to Falkow et al), WO-A-88/01302 (published Feb. 25, 1988) and references mentioned therein!.
Among nucleic acid probe assays are what are known in the art as "sandwich" assays in which two probes are used to sandwich the nucleic acid of interest therebetween in a three-part hybridized product. Generally, one probe is a "capture" probe which is immobilized or capable of becoming so on a solid surface, and the other probe is detectably labeled or capable of becoming so. Sandwich assays have the advantage that the predetermined nucleic acid need not be immobilized directly to a solid support, and offer the potential for higher specificity because two hybridization reactions are required for detection instead of one.
Most "capture" probes used in probe assays are generally composed of a sequence of nucleotides which form an oligonucleotide which is complementary to at least one nucleic acid sequence of the predetermined nucleic acid being detected. Various methods are known for attaching oligonucleotides to solid supports for affinity chromatography separation of nucleic acids and for probe assays. Among the considerable literature describing such methods are WO-A-88/01302 (noted above), EP-A-0 235 726 (published Sep. 9, 1987) and U.S. Pat. No. 4,673,657 (issued Jun. 16, 1987 to Christian).
A significant advance in the art is described in U.S. Pat. No. 4,683,195 (issued Jul. 28, 1987 to Mullis et al) and U.S. Pat. No. 4,683,202 (issued Jul. 28, 1987 to Mullis). Without going into extensive detail, these patents describe an amplification process wherein primers are hybridized to nucleic acid templates in the presence of a polymerization agent (such as a polymerase) and four deoxyribonucleoside triphosphates, and extension products are formed from the primers. These products are denatured and used as templates in a cycling reaction which amplifies the number and amount of predetermined nucleic acid to facilitate its subsequent detection. This amplification process can be carried out cyclically as many times as desired to produce a larger quantity of detectable material from a small amount of predetermined nucleic acid.
Once the target sequence has been adequately amplified to detectable quantities, the mode of detection is not critical. Many techniques for detection are described in the art including the use of probes labeled with radioisotopes, biotin or enzymes (linked to the probe through a biotin-avidin linkage) or gel electrophoresis. Other probes are used for capturing the amplified product on a support.
U.S. Pat. No. 4,727,019 (Feb. 23, 1988 to Valkirs et al) describes analytical methods and apparatus in which nucleic acids can be detected using probes immobilized directly to a porous substrate (such as a membrane) in a localized region. Alternatively, the probe can be embedded within the porous matrix. While directly attaching probes to such substrates or embedding them therein may provide accurate and sensitive assays where the predetermined nucleic acid is present in the test specimen in generally large concentrations, it has limitations where the concentrations are very low. As research proceeds in this field of art, the need to detect lower quantities (even a single molecule) is of greater importance. Thus, many conventional methods and apparatus for nucleic acid testing are deficient.
Similarly, an analytical method is described in EP-A-0 200 381 (published Nov. 5, 1986) which utilizes nucleic acids attached to polymeric particles which are embedded within a porous matrix. Moreover, several probes can be embedded in distinct regions of the matrix so that a multiplicity of nucleic acids can be detected simultaneously.
The desire to detect one or more nucleic acids simultaneously remains in the art. However, as noted above, there is also a need to detect increasingly lower concentrations of those acids. This requires high sensitivity by the probes and analytical procedures.